The overall objective of this proposal is to continue our investigations into the roles of soluble and membrane Kit-ligand (KL) in vivo and the mechanism of the formation of soluble KL in vivo and in vitro. Membrane growth factors which are processed to produce soluble ligands may function both as soluble factors and as membrane factors. Important questions concerning membrane growth factors include the role of soluble and membrane forms in vivo and the elucidation of mechanisms governing their expression. The membrane growth factor Kit-Ligand (KL), the ligand of the Kit receptor tyrosine kinase, is encoded at the Sl locus and mice carrying SIS mutations have defects in hematopoiesis, gametogenesis and melanogenesis. Two alternatively spliced KL transcripts encode two cell associated KL protein products, KL-1 and KL-2. The KL-2 protein lacks the major proteolytic cleavage site for the generation of soluble KL, thus representing a more stable cell-associated form of KL. In this application in Specific Aim 1 we propose to investigate the regulation of KL-1 and KL- 2 mRNA expression in vivo during embryogenesis and in the postnatal animal by tagging KL-1 and KL-2 mRNAs using a novel knock-in strategy. In Specific Aim 2 we will continue our investigation of the in vivo role of membrane and soluble KL using knock-in mouse models. A) For this purpose mice expressing a membrane obligatory KL-2 protein by knock-in technology will be made and phenotypes of heterozygous and homozygous mutant mice will be characterized with particular emphasis on the determination of the dynamics of hematopoietic cell populations in mutant mice. B) By using knock-in technology mice expressing KL-1 exclusively will be obtained and the phenotypes of hetero-and homozygous mutant mice will be characterized. Specific Aim 3 addresses the role of cytoplasmic domain sequences of KL in KL/Kit function. In these studies we will examine the consequences, in vivo, a) of replacement of cytoplasmic domain sequences with those of CSF- 1 and b) of deletion of cytoplasmic domain sequences of KL by using knock- in strategy. Furthermore we propose to use fetal liver derived stromal cell lines capable of supporting long-term hematopoiesis in vitro to investigate role of cytoplasmic domain using cell biological approaches. Specific Aim 4 focuses on the elucidation of the mechanism of proteolytic processing of KL-1 and KL-2 and Kit receptor products. In these studies we will evaluate in vitro KL-1, KL-2 and Kit peptides as target sequences for proteolytic cleavage by disintegrins TACE, MDC9 and MDC15. Furthermore, in order to evaluate the in vivo role of disintegrins in the production of soluble KL and Kit in serum knock-out mouse models will be used.